Inhibition of a nutritional endosymbiont by glyphosate abolishes mutualistic benefit on cuticle synthesis in Oryzaephilus surinamensis.

Glyphosate is widely used as a herbicide, but recent studies begin to reveal its detrimental side effects on animals by targeting the shikimate pathway of associated gut microorganisms. However, its impact on nutritional endosymbionts in insects remains poorly understood. Here, we sequenced the tiny, shikimate pathway encoding symbiont genome of the sawtoothed grain beetle Oryzaephilus surinamensis. Decreased titers of the aromatic amino acid tyrosine in symbiont-depleted beetles underscore the symbionts' ability to synthesize prephenate as the precursor for host tyrosine synthesis and its importance for cuticle sclerotization and melanization. Glyphosate exposure inhibited symbiont establishment during host development and abolished the mutualistic benefit on cuticle synthesis in adults, which could be partially rescued by dietary tyrosine supplementation. Furthermore, phylogenetic analyses indicate that the shikimate pathways of many nutritional endosymbionts likewise contain a glyphosate sensitive 5-enolpyruvylshikimate-3-phosphate synthase. These findings highlight the importance of symbiont-mediated tyrosine supplementation for cuticle biosynthesis in insects, but also paint an alarming scenario regarding the use of glyphosate in light of recent declines in insect populations.

Bacteriome-Associated Endosymbiotic Bacteria of Nosodendron Tree Sap Beetles (Coleoptera: Nosodendridae).

The family Nosodendridae is a small group of tree sap beetles with only 91 described species representing three genera from the world. In 1930s, bacteria-harboring symbiotic organs, called bacteriomes, were briefly described in a European species Nosodendron fasciculare. Since then, however, no studies have been conducted on the nosodendrid endosymbiosis for decades. Here we investigated the bacteriomes and the endosymbiotic bacteria of Nosodendron coenosum and Nosodendron asiaticum using molecular phylogenetic and histological approaches. In adults and larvae, a pair of slender bacteriomes were found along both sides of the midgut. The bacteriomes consisted of large bacteriocytes at the center and flat sheath cells on the surface. Fluorescence in situ hybridization detected preferential localization of the endosymbiotic bacteria in the cytoplasm of the bacteriocytes. In reproductive adult females, the endosymbiotic bacteria were also detected at the infection zone in the ovarioles and on the surface of growing oocytes, indicating vertical symbiont transmission via ovarial passage. Transmission electron microscopy unveiled bizarre structural features of the bacteriocytes, whose cytoplasm exhibited degenerate cytology with deformed endosymbiont cells. Molecular phylogenetic analysis revealed that the nosodendrid endosymbionts formed a distinct clade in the Bacteroidetes. The nosodendrid endosymbionts were the most closely related to the bacteriome endosymbionts of bostrichid powderpost beetles and also allied to the bacteriome endosymbionts of silvanid grain beetles, uncovering an unexpected endosymbiont relationship across the unrelated beetle families Nosodendridae, Bostrichidae and Silvanidae. Host-symbiont co-evolution and presumable biological roles of the endosymbiotic bacteria are discussed.

A Novel, Extremely Elongated, and Endocellular Bacterial Symbiont Supports Cuticle Formation of a Grain Pest Beetle.

The saw-toothed grain beetle, Oryzaephilus surinamensis (Silvanidae), is a cosmopolitan stored-product pest. Early studies on O. surinamensis in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of O. surinamensis in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle, Rhyzopertha dominica (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of O. surinamensis is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest.IMPORTANCE Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, Oryzaephilus surinamensis, in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution.